The objective is to isolate the enzymes involved in the total synthesis of acetate from CO2 by the heterotrophic bacteria, Clostridium thermoaceticum and Clostridium acidiurici and study the mechanism of catalysis by the enzymes. The synthesis by C. thermoaceticum is unique in that it involves the carboxylation of a methyl group attached to a cobalt of a corrinoid enzyme and the carboxylation occurs by transcarboxylation from pyruvate. This system is studied using 14CH3-tetrahydrofolate and pyruvate as the substrates and the assay consists of determination of the 14C converted to acetate. In the fermentation proper, CO2 enters the CH3-THF by a reduction of CO2 to formate, formate is converted to formyl-THF, which is reduced to CH3-THF. CO2 exchanges with the pyruvate carboxyl. Thus, acetate is formed in which both carbons are derived from CO2. We now propose to purify the transcarboxylase and study the mechanism of the conversion of CH-THF and pyruvate to acetate. With C. acidiurici, we have found that the synthesis of acetate from CO2 does not occur by the mechanism used by C. acidiurici. In this case, it appears that glycine is synthesized from CH2 double bond THF, CO2 and NH3. A second CH2 double bond THF then combines with the glycine forming serine. The serine is coverted via pyruvate to acetate. Since CH2 double bond THF is formed from CO2 by reduction to formate which is converted to formyl-THF and reduced to CH2 double bond THF, both carbons of acetate arise from CO2. The key enzyme in this mechanism is glycine decarboxylase. This enzyme has not been isolated from C. acidiurici. We propose to isolate the enzyme and study the mechanism of the reaction. Both of these pathways offer an opportunity to study reactions which are unique in fermentations.